Carbon Amendments Shape the Bacterial Community Structure in Salinized Farmland Soil

ABSTRACT Practical, effective, and economically feasible salt reclamation and amelioration methods are in great demand in arid and semiarid areas. Energy amendments may be more appropriate than alternatives for improving salinized farmland soil because of their effects on soil microbes. We investigated the effects of biochar (Carbon) addition and desulfurization (noncarbon) on the soil bacterial community associated with Zea mays seedlings. Proteobacteria, Firmicutes, and Actinobacteriota were the dominant soil bacterial phyla. Biochar significantly increased soil bacterial biodiversity but desulfurization did not. The application of both amendments stimulated a soil bacterial community shift, and biochar amendments relieved selection pressure and increased the stochasticity of community assembly of bacterial communities. We concluded that biochar amendment can improve plant salt resistance by increasing the abundance of bacteria associated with photosynthetic processes and alter bacterial species involved in carbon cycle functions to reduce the toxicity of soil salinity to plants. IMPORTANCE Farmland application of soil amendments is a usual method to mitigate soil salinization. Most studies have concluded that soil properties can be improved by soil amendment, which indirectly affects the soil microbial community structures. In this study, we applied carbon and noncarbon soil amendments and analyzed the differences between them on the soil microbial community. We found that carbon soil amendment distinctly altered the soil microbial community. This finding provides key theoretical and technical support for using soil amendments in the future.

This paper reports on an experiment in which different laboratory soil mesocosms that used relatively saline soils to which biochar and gypsum additions were done to test what effects these substances might have on basic plant physiology parameters and changes in the soil microbiome. There is significant interest in the use of biochar as a soil amendment to improve soil health and potentially improve C-uptake and sequestration within soils; however experimental evidence for the role of biochar is generally lacking, thus this work has the potential to aid our understanding of its role in these processes.
The paper as written is difficult to follow. Principally, the authors refer to biochar as an 'energy substrate', but provide no evidence that biochar itself is used as a microbial energy source, either electron acceptor or donor, directly. The experimental system itself is a highly contrived single laboratory experiment, thus field relevance is harder to interpret, and experimental details are missing. The interpretation of the data is, in places, speculative to the point that basic ecological principles are difficult to follow, in part because this is entirely a 16S amplicon based community study. Some further specifics are given below.
Specific comments. The title is uninformative as written since it's not clear what 'energy amendments' are.
l96, Unclear what is meant by 'pots were poured'? Does this mean 200ml of water added? What's the total volume of soil? What was the moisture content before and after addition.
l. 100. Biochar is simply described as maize, 400C, this is inadequate. Biochar itself is a complex compound, and the specific conditions under which it is produced can influence it's properties, thus significantly more detail is need here. Furthermore, the amounts of addition and % in soil are not given for any of the additions. l104/5 What was the original salt concentration of these soils.
P12. It's not clear what 'modified stochastic ratio' is or means, nor it is explained in the text. My general interpretation of the results is that there is a modest statistically significant impact of the biochar treatment on the microbial community, while other treatments were not significant. Thus, it's possible that the biochar may have, to some extent, selected for a different community, which would be determined by some, not identified here, property that the microbes are responding to. It's not clear to me how this is 'stochastic'. Furthermore, without more experimental details of how amendment concentrations and how these effect total soil moisture and organic C contents, it's a bit difficult to fully interpret. lower part pg 14 & pg15, A number of studies are cited, however it's unclear how this work directly links to these studies and adds something new.
P15 The last statement of the MS about effects of biochar is largely conjecture. This experiment did not directly test nutrient mobilization or microbial growth as a result of biochar amendment to lend direct support to these ideas, since the microbes identified as significant have a variety of metabolisms/responses to environmental conditions. Reviewer #2 (Comments for the Author): • The "much easier" in the title is a bit awkward; consider revising (e.g. energy amendments shape the bacterial community structure in salinized farmland soil more than..." • Line 18 -associated (add "d") • Line 20 -phyla (plural); Delete "And" in the next sentence • Line 23 -the phrase "drive the bacterial communities more stochastic" is a bit confusing and unclear; perhaps the form of "stochastic" needs to be changed; what properties of the bacterial communities are being driven? Is it structural changes? Shifts in abundance? Community assembly? • Line 24 -the usage of "however" is inappropriate here • Line 25 -the usage of the word "conducive" seems out of place; perhaps the authors mean "effective" or "influential"? • Line 25 -the phrase "soil functional bacterial community species composition" -does this mean functional diversity or species diversity or relative abundance? • Line 35 -is this "further" or "future"? • Line 44 -revise "intensifies" • Line 69 -is "infect" really what the authors mean? Or is it "affect"? • Line 78 -"maintain" (remove "s"); "enable" (remove "s") • Line 79 -"tolerate" (remove "s") • Line 95 -what soil type? • Manuscript may need language editing to correct some minor errors • Randomization of pots? CRD? Were photosynthetic parameters measured for all leaves per plant? • At what age and stage of the plant were the soil samples collected? This needs to be specified because bacterial community structure changes with the age of the plant. Processes that determine community assemblage also change with host plant age. • Line 186 -is it 4 functions or just 3? C-cycle, energy, and N-cycle • Line 191 -the plural of index is indices • Line 365-369 -please check if the statement is correct (BA is not mentioned while DA is mentioned twice) • Line 382 -"change in bacterial composition of soil had more effects on plant traits than the change of abundances" -this statement seems contradictory to the previous statement that increasing Proteobacteria abundance in RS under BA significantly enhanced PS • What's the significance of the observed changes in the co-occurrence networks? How does this relate to the issue of soil salinization? • What does the increased influence of stochastic processes on microbial community assemblage upon amendment of Biochar mean in relation to the problem of soil salinization? Will this effect be carried over until harvest? Or is the increased stochasticity observed only due to the short span of time since application of biochar? • How does the study address the 3rd objective -To assess whether the change of soil bacterial community could affect the ability of host plants' salt resistance? How was host plant salt resistance evaluated? Is this through photosynthetic rate? Does high Ps rate automatically mean salt resistance?
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Dear Editors and Reviewers:
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate the editor and reviewers very much for their constructive comments and suggestions on our manuscript entitled "Energy amendments much easier shape the bacterial community structure in salinized farmland soil" (control no.

Spectrum01012-22).
We have studied reviewers' comments carefully. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. According to the reviewers' detailed Under your suggestion, we changed the title to: Carbon amendments shape the bacterial community structure in salinized farmland soil differ from non-carbon.
l96, Unclear what is meant by 'pots were poured'? Does this mean 200ml of water added? What's the total volume of soil? What was the moisture content before and after addition.
We apologize for the oversight and misunderstanding. 'pots were poured' should be 'Pots were irrigated with 200 ml distilled water'. We have now provided additional methodological details for the soil. The experimental soils were collected from an agricultural field cultivated with maize in the Aral District, Xinjiang Province, in northern China. The soil was air-dried, ground, and sifted through a 5mm sieve before the experiment (Tables S1). The experimental design in included four treatments: 500g soil per pot (CK), 475g soil + 25g desulfurized gypsum addition per pot (DA), 485g soil + 15g biochar addition per pot (BA), and 460g soil + 15g biochar addition + 25g desulfurized gypsum addition per pot (DBA). Six replicates were maintained per treatment. We have added above information in the revised manuscript. Line 90-104.
l. 100. Biochar is simply described as maize, 400C, this is inadequate. Biochar itself is a complex compound, and the specific conditions under which it is produced can influence it's properties, thus significantly more detail is need here. Furthermore, the amounts of addition and % in soil are not given for any of the additions.
Thank you for your suggestion. We have added information about biochar in the revised manuscript and Supplementary  Supplementary Table S1. P12. It's not clear what 'modified stochastic ratio' is or means, nor it is explained in the text. My general interpretation of the results is that there is a modest statistically significant impact of the biochar treatment on the microbial community, while other treatments were not significant. Thus, it's possible that the biochar may have, to some extent, selected for a different community, which would be determined by some, not identified here, property that the microbes are responding to. It's not clear to me how this is 'stochastic'. Furthermore, without more experimental details of how amendment concentrations and how these effect total soil moisture and organic C contents, it's a bit difficult to fully interpret.
We thank for your comment and we sorry that we did not write this point clearly in the originally submitted manuscript. Deterministic assembly processes can result from either homogenous or variable selection with respect to environmental characteristics (Barnett et al. 2020). In this study, we considered that the process of soil bacterial community assembly should be deterministic in four treatments. But the MST values of the BA and DBA were significantly higher than CK and DA. Upon reviewing the literature, we found that soil organic carbon was the major drivers of bacterial β diversity and bacteria were more influenced by undominated-based stochastic processes (Chen et al. 2022). And the stochastic process of bacterial community assembly was influenced by soil carbon metabolism. Therefore, after adding the biochar with high organic carbon content, there is a transition in soil bacterial community assembly process from homogeneous selection to stochasticity. As your Thank you for the above suggestion. We have removed the last statement and re-written the discussion section to include a more comprehensive view of the change of soil bacterial community could affect the ability of host plants' salt resistance. Line

398-427.
Thank you for reading our paper carefully and giving the above positive comments.
We appreciate your warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer #2 (Comments for the Author):
Dear reviewer2: I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in manuscript. According your advice, we have thoroughly checked and polished the English of our manuscript with the help of a native English speaker John Wilmshurst. Some of your questions were answered below: •The "much easier" in the title is a bit awkward; consider revising (e.g. energy amendments shape the bacterial community structure in salinized farmland soil more than..." According to your advice, we agree and changed the title to "Carbon amendments shape the bacterial community structure in salinized farmland soil differ from non-carbon" • Line 18 -associated (add "d") Thank for your suggestion, we have made it.
• Line 20 -phyla (plural); Delete "And" in the next sentence We have modified it.
• Line 23 -the phrase "drive the bacterial communities more stochastic" is a bit confusing and unclear; perhaps the form of "stochastic" needs to be changed; what properties of the bacterial communities are being driven? Is it structural changes? Shifts in abundance? Community assembly?
Thank you for your suggestion. We revised the expression of this point as follow: amendments would relieve selection pressure and increase the stochasticity of community assembly of bacterial communities. Line 22-26.
• Line 24 -the usage of "however" is inappropriate here We have modified it.
• Line 25 -the usage of the word "conducive" seems out of place; perhaps the authors mean "effective" or "influential"?
We have revised it according to your advice.
• Line 25 -the phrase "soil functional bacterial community species composition"does this mean functional diversity or species diversity or relative abundance? Yes, it means the relative abundance of soil functional bacterial species for "C-cycle", "Energy" and "N-cycle". We have modified it in the revised manuscript.
We are very sorry for this mistake. We have modified it.
• Line 69 -is "infect" really what the authors mean? Or is it "affect"?
We are very sorry for this mistake. We have modified it.
• Line 79 -"tolerate" (remove "s") We are very sorry for this mistake. We have modified it.
Thank you for your advice. The soil type is classified as sandy loam. We have added this information in the revised manuscript.
• Manuscript may need language editing to correct some minor errors According your advice, we have thoroughly checked and polished the English of our manuscript with the help of a native English speaker John Wilmshurst.
•Randomization of pots? CRD? Were photosynthetic parameters measured for all leaves per plant?
Thank you for pointing out this statement. To make it clear, we added the sentence in Line 109-113 to the following:" After 20 days of seedling growth (two leaves stage), three pots were randomly selected in each treatment to determine the photosynthesis and leaf water potential of seedlings and collect soil samples for microbial analysis.
Measurements of photosynthesis were conducted using youngest fully expanded main-stem leaf (the 1st leaf from the apex)." • At what age and stage of the plant were the soil samples collected? This needs to be specified because bacterial community structure changes with the age of the plant.
Processes that determine community assemblage also change with host plant age.
Thank you for pointing to the missing information. In this study, we want to improve the farmland abandonment due to the soil salinity by use of amendments. Hence, the experimental soils were collected from farmland abandonment where previous cultivated maize in the Aral District, Xinjiang Province, in northern China. We have added the above information in the revised manuscript. Line 90-94.
• Line 186 -is it 4 functions or just 3? C-cycle, energy, and N-cycle We are very sorry for this mistake. We have modified it.

• Line 191 -the plural of index is indices
We are very sorry for this mistake. We have modified it.
• Line 365-369 -please check if the statement is correct (BA is not mentioned while DA is mentioned twice) We are very sorry for this mistake. We have modified it in the revised manuscript.
• Line 382 -"change in bacterial composition of soil had more effects on plant traits than the change of abundances" -this statement seems contradictory to the previous statement that increasing Proteobacteria abundance in RS under BA significantly enhanced PS Thank you for your suggestion. We have re-written this part in the revised manuscript (Line 397-427) to remove the ambiguity.
• What's the significance of the observed changes in the co-occurrence networks?
How does this relate to the issue of soil salinization?
Thanks very much for you question. First, we found that the complexity of bacterial networks increased with the application of BA and DA, respectively. This means that the soil microbial communities were more resistant to salt stress relative to CK treatment. Second, different soil amendments could increase the interactions of different bacterial phyla. AND adding the biochar was able to enhance the interaction among the Proteobacteria phyla, while desulfurization was the Firmicutes phyla.
Proteobacteria and Firmicutes were reported as "good indicators" for reflecting changes of the main microbial groups and important resource for exploring halophilic enzymes and metabolic pathways for pollutant remediation in saline soil. We have re-written this part in the revised manuscript. Line 362-388.
• What does the increased influence of stochastic processes on microbial community assemblage upon amendment of Biochar mean in relation to the problem of soil salinization? Will this effect be carried over until harvest? Or is the increased stochasticity observed only due to the short span of time since application of biochar?
Thank you very much for your good question. We generally considered that Soil properties among treatments in this study are broadly consistent, so the bacterial community in the soil should be the same development process. In our study, we found that stochastic processes dominated the soil bacterial community assembly only with biochar addition. Some studies have demonstrated increased resource supply can increase stochasticity under physicochemical conditions that do not impose strong selection (Dini-Andreote et al., 2015). Hence, we suppose that the increasing carbon-related bacterial species would enhance the function and role of soil microbial communities after adding the biochar, which may enhance the function and role of soil microbial communities to develop resistance to soil salinity stress. And we suggest that any addition of soil carbon amendment would increase the stochastic process of soil bacterial community assembly. We have added above sentences in the revised manuscript. Line 347-361.
• How does the study address the 3rd objective -To assess whether the change of soil bacterial community could affect the ability of host plants' salt resistance? How was host plant salt resistance evaluated? Is this through photosynthetic rate? Does high Ps rate automatically mean salt resistance?
Thank you for the above suggestion. We have re-written the discussion section to include a more comprehensive view of the change of soil bacterial community could affect the ability of host plants' salt resistance. Line 398-427.
Thank you for reading our paper carefully and giving the above positive comments.
We appreciate your warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions. Thank you for submitting your revised manuscript to Microbiology Spectrum. I can now inform you that the Editorial Board has evaluated the manuscript Spectrum01012-22R1: Energy amendments much easier shape the bacterial community structure in salinized farmland soil.
The manuscript has been reviewed carefully by experts (comments given below). The Editor has advised that the manuscript will be considered for publication after minor revision.
Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
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Editor, Microbiology Spectrum Reviewer comments: Reviewer #2 (Comments for the Author): Thank you very much; all my comments have been addressed satisfactorily. One last suggestion, though --I suggest that the title be revised. Maybe you can consider the following: "Carbon amendments shape the bacterial community structure in salinized farmland soil differently compared to non-carbon treatment" or simply "Carbon amendments shape the bacterial community structure in salinized farmland soil"

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Thank you for submitting your paper to Microbiology Spectrum.

Dear Editors and Reviewers:
Thank you very much for giving us an opportunity to revise our manuscript. We appreciate the editor and reviewers very much for their constructive comments and suggestions on our manuscript entitled "Carbon amendments shape the bacterial community structure in salinized farmland soil differ from non-carbon "(Spectrum01012-22R2).
We have studied reviewers' comments carefully. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. According to the reviewers' detailed suggestions, we have made a careful revision in "Marked-Up Manuscript". Revised portion are marked in the manuscript (tracked changes). We hope our revised manuscript can be accepted for publication. The main corrections in the paper and the responds to the reviewer's comments are as flowing:

Dr. Qisheng Han
On behalf of all authors.

Reviewer #2
Dear reviewer2, I am very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in manuscript. Revised portion are marked in the manuscript (tracked changes). Here are responses to your comments: 1. Maybe you can consider the following: "Carbon amendments shape the bacterial community structure in salinized farmland soil differently compared to non-carbon treatment" or simply "Carbon amendments shape the bacterial community structure in salinized farmland soil"